The present invention relates to new agglutination tests for detecting influenza viruses and to reagents for carrying out these tests.
Influenza, a contagious, endemic and epidemic illness, practically the only treatment for which is vaccination, poses serious prophylactic problems because of the antigenic variability of the influenza viruses. Generally, the detection of influenza viruses and the resulting diagnosis of influenza are important for three essential reasons:
in the epidemiologic sphere in keeping a check on the degree of immunity of populations during an influenza epidemic;
in the preventative field, for identifying strains to be introduced into the vaccines;
in the clinical field, finally, for the accurate diagnosis of the influenza infection, in particular for the abnormal and rare clinical forms.
This is why techniques have been proposed for some years for detecting the influenza virus. These techniques may be classed into two main categories, biological techniques and immunological techniques, these latter comprising the conventional hemagglutination techniques and techniques for measuring the titre of antibodies capable of inhibiting the activity of the neuraminidase. The biological technique which consists in isolating and identifying the virus itself after culture in embryonated eggs or on monkey kidney cells is long and requires a highly-qualified staff. On the other hand, the immunological techniques are easy to implement and so are more widely used than the biological technique, in particular the hemagglutination inhibition method. Hemagglutination is visible only in the case where the serum analyzed does not contain non specific antibodies which mask the hemagglutinating sites. However, these immunological techniques may present difficulties which are sometimes difficult to overcome, namely:
the presence of non-specific inhibitors,
variations in the biological and antigenic characteristics of the viruses,
variations in the quality of the erythrocytes.
On the other hand, the inhibition of the enzymatic activity of the neuraminidase (Mucopolysaccharide N-acetylneuraminylhydrolase of the influenza viruses; international classification 3.2.1.18) is a very good method for detecting the specific anti-neuraminidase antibodies of a strain of the influenza virus. The neuraminidase is identified by inhibition of the activity of the enzyme by means of an anti-serum prepared against the reference virus antigens. The amount of influenza virus is measured by determining the amount of neuraminidase (compare in particular the work of P. CUATRECASAS and G. ILLIANO in Biochem. and Biophys. Res. Communications (1971), 44, 1, 178-184). The determination of the neuraminidase comprises two principal steps:
(a) the step of incubating the virus alone or the virus pretreated by means of the anti-serum, with fetuin--the substrate of the neuraminidase--for 18 hours at 37.degree. C.;
(b) the step of determination of the sialic acid released by the action of the neuraminidase, by means of the technique of WARREN (L. WARREN, J. Biol. Chem. 234 (1959) 1971). This latter determination is very long and complicated and comprises several steps which require numerous reagents, accurate heating times and finally colorimetric measurements after extraction of the thiobarbituric derivative of the oxidized sialic acid in ButOH/HCl. The routine determination of several samples is then particularly laborious for determination of sialic acid with a view to detecting the neuraminidase, all the following Articles: Journal of Biological Standardization 1976 4, 225-241 O. THAENHART and E. K. KUWERT and Biochimica et Biophysica Acta, 523 (1978) 435-442 U. V. SANTER, J. YEE-FOON and M. C. GLICK).
The aim of the present invention is accordingly to provide a new test for detecting influenza viruses which combines the specificity of the enzymatic reaction with agglutination techniques. It is easy to carry out and gets over the disadvantages of these two methods taken separately.